Proline Metabolism IncreaseskatGExpression and Oxidative Stress Resistance in Escherichia coli

Abstract

The oxidation of L-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and ⌬ 1 -pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type and putA mutant strains of Escherichia coli. Initial stress assays revealed that the putA mutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in the putA mutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded by katG) expression and activity. Furthermore, the ⌬katG strain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression of katG along with several other genes involved in oxidative stress defense. In addition to katG, proline increased the expression of grxA (glutaredoxin 1) and trxC (thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance in E. coli via a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity.T he conversion of L-proline to glutamate is a four-electron oxidation process that is coordinated in two successive steps by the enzymes proline dehydrogenase (PRODH) and ⌬ 1 -pyrroline-5-carboxylate dehydrogenase (P5CDH) (Fig. 1) (1). In eukaryotes, PRODH and P5CDH are separately encoded enzymes localized in the mitochondrion. In Gram-negative bacteria, PRODH and P5CDH are combined into a bifunctional enzyme known as proline utilization A (PutA) (1, 2). The PRODH domain contains a noncovalently bound flavin adenine dinucleotide (FAD) cofactor and couples the two-electron oxidation of proline to the reduction of ubiquinone in the cytoplasmic membrane (3). The product of the PRODH reaction, ⌬ 1 -pyrroline-5-carboxylate (P5C), is subsequently hydrolyzed to glutamate-␥-semialdehyde (GSA), which is then oxidized to glutamate by the NAD ϩ -dependent P5CDH domain (2). In certain Gram-negative bacteria such as Escherichia coli, PutA also has an N-terminal ribbon-helix-helix (RHH) DNA-binding domain (residues 1 to 47) (4). The RHH domain enables PutA to act as an autogenous transcriptional regulator of the putA and putP (highaffinity Na ϩ -proline transporter) genes (4). PutA represses put gene expression by binding to five operator sites in the put regulatory region (5). Transcription of the put genes is activated by proline, which causes a r...

Proline utilization system is required for infection by the pathogenic α-proteobacterium Brucella abortus

“…abortus DputR is hypersensitive to oxidative stress in vitro It has been shown previously that bacterial Put systems are linked to oxidative stress [28][29][30]. To test the hypothesis that putR and putA in B. abortus are required for the ability of the bacteria to cope with oxidative stress, we cultivated the bacteria in liquid medium, and then the cultures were treated with hydrogen peroxide (H 2 O 2 ) at a final concentration of 10 mM.…”

“…It has also been shown that the PutA acts to induce anti-oxidative stress genes in E. coli through production of reactive oxygen species (ROS) [28,30]. The exact mechanism for this ROS production was not explored, but it was suggested that ROS were generated through the increased flux through the electron transport chain, leading to the formation of superoxide species and production of H 2 O 2 .…”

Gold‐Ions‐Mediated Diproline Peptide Nanocarpets and Their Inhibition of Bacterial Growth

“…The variation in the physiological state of S. Aureus , B. subtilis and E. coli , cells in Luria Lertani (LB) broth varies greatly with time . The addition of Pro‐Pro increased the growth of these bacteria, thus revealing that this peptide is also stimulating the growth of bacteria, perhaps by acting as a proteogenic molecule for them ,. However in the presence of the Au(III)‐conjugated Pro‐Pro, the growth was altered and decreased (ESI).…”

The metabolic pathways utilized by Salmonella Typhimurium during infection of host cells

“…Proline biosynthesis has been linked to resistance to oxidative stress through the proline catabolic pathways involving the bifunctional flavoenzyme, PutA, which is both a proline dehydrogenase (generates P5C) and Δ 1 ‐pyrroline‐5‐carboxylate (P5C) dehydrogenase (generates glutamate). Furthermore, proline catabolism involving PutA was found to be involved in the generation of hydrogen peroxide (Zhang et al ., ). These observations suggest it is not a defect in the biosynthesis of proline per se , but rather the accumulation of the intermediate metabolite, P5C, which is responsible for the intracellular growth defects.…”